1. Field of the Invention
The present invention relates to a method for measuring quantitatively sugar-alcohols, a column and a kit for the process.
2. Description of the Prior Art
Sugar-alcohols substances such as 1,5-anhydroglucitol (referred to as AG hereafter) and myo-inositol (referred to as MI hereafter) have been receiving attention in recent years as markers for the diagnosis of diabetes mellitus.
In quantitative analysis of sugar-alcohols in human body fluid, there have been used extracts which have been freed of interfering substances such as saccharides (particularly, glucose) and proteins which are generally contained abundantly in the human biological fluid and which have an influence on the analysis.
Removal of the aforementioned interfering substances, particularly proteins has required heretofore centrifugal separation, and is troublesome. Especially, taking account of clinical applications, simplification of the removal operation has been desired. It is preferred for quantitative analysis of sugar-alcohols in a number of test samples to remove simultaneously saccharides and proteins.
Techniques for determining sugar-alcohols in blood have been heretofore proposed, including the measurement of protein-freed serum or plasma, after converting them to derivatives, on gas chromatography (abbreviated to a GC method hereafter), or the measurement of AG in effluents from an interfering substance-removing column, through which protein-freed test samples have been passed, using an enzyme colorimeter (abbreviated to an enzyme colorimetric method hereafter) (EP 261591-A).
The GC method involves a protein-removing step and a derivative-producing step before subjecting the sample to gas chromatography, and requires several column chromatographic operations and condensing to dryness operations so that it requires many handlings, is time consuming and is unsuitable for measuring a number of test samples. Depending upon the samples, there are cases where undeterminable peaks overlap with sugar-alcohol peaks rendering the resulting measurements inaccurate.
The enzyme colorimetric method comprises passing protein-freed samples through an interfering substance-removing column to remove the interfering substances, primarily saccharides, and oxidizing AG with oxygen simultaneously producing hydrogen peroxide, an amount of which is quantitatively determined after coloration with enzyme by a colorimetric measurement. Comparing with the GC method, it has fewer steps, and is capable of treating a large number of samples so that it may be regarded as an excellent method. For clinical examination, however, it involves some time-consuming troublesome steps requiring many handlings with the coloration step taking a long time to reach a stable state, so that the number of samples measurable by one operator is limited. Either one of the aforementioned two methods is very time-consuming with many handlings in determination of sugar-alcohols, and can not be easily conducted for the measurement.
In order to overcome the difficulties as described above, the present inventors have made a research on a method for automating the measurement of sugar-alcohols, and discovered that even when samples of serum or blood plasma are injected sequentially into an interfering substance-removing column, through which water or the like is allowed to steadly flow, as an apparatus for directly measuring them, the interfering substances can be completely removed to permit determination of the sugar-alcohols. Moreover, we have made a column and a kit for use in the measurement. The present invention has been accomplished on the basis of the above discoveries.